KR102240417B1 - Anti-transthyretin human antibody - Google Patents

Abstract

A human antibody comprising a complementarity determining region of an H chain consisting of the amino acid sequence of SEQ ID NOs: 1 to 3, and a complementarity determining region of an L chain consisting of the amino acid sequence of SEQ ID NOs: 4 to 6. The human antibody of the present invention has an activity that specifically binds to transthyretin (TTR) that caused a structural change or an activity to inhibit fibrosis of TTR, and is a human antibody suitable for application to the human body.

Description

Anti-transtyretin human antibody {ANTI-TRANSTHYRETIN HUMAN ANTIBODY}

The present invention provides an antibody that effectively inhibits the formation of amyloid fibers or deposition of transthyretin (TTR) into tissues, and a therapy using the antibody. This antibody therapy is based on a new treatment strategy that does not act on normal TTR, but inhibits only amyloid formation of ideal TTR, and is expected to be a novel treatment with excellent safety.

Amyloidosis is a group of diseases in which dysfunction is caused by depositing proteins forming a fibrous structure in organs of the whole body, and various diseases such as Alzheimer's type dementia and prion's disease are included (Non-Patent Document 1).

Familial Amyloidotic Polyneuropathy (FAP) is an autosomal dominant hereditary systemic amyloidosis caused by a point mutation or deletion of genes such as TTR, apolipoprotein A1, and gelsolin (non-patent literature 2). Among them, FAP is the most common due to genetic mutations in TTR. It is known that mutant TTR forms amyloid fibers, and usually deposits in peripheral nerves, heart, kidney, digestive tract, eyes, brain, meninges, and other tissues of the entire body to cause dysfunction. The patient's prognosis is very poor, and it is an incurable disease ranging from onset to death in about 10 years.

To date, point mutations or deletions of more than 100 TTR genes have been reported. Among them, the Val30Met mutation (hereinafter referred to as “V30M”) in which valine at the 30th TTR is mutated to methionine is the most common, and the number of patients is in Portugal, Sweden, and Japan. More than 6,000 FAP patients are confirmed in Portugal, and there are a number of regions where FAP is not considered to be investigated, and the FAP patient group is expected to continue to be discovered worldwide, which is enough to exceed 10,000 in the world. It is thought that there are patients who do. From recent studies, it has been found that the clinical picture of FAP (age onset, specificity of organs deposited, etc.) is greatly influenced by the type of mutation of the TTR gene (Non-Patent Document 3). For example, with regard to the onset age of FAP, the L55P mutation causes a severe clinical picture of onset in teens, but the V122I mutation occurs after the age of 60. On the other hand, in the V30M mutation, it is known that both a disease type that develops in young adults and a disease type that develops in old age exist. Regarding the specificity of the organs to be deposited, the D18G mutation deposits in the brain or meninges and causes central nervous disorders, while the V30M mutation deposits in the systemic tissues and causes peripheral nerve disorders and myocardial disorders (Non-Patent Document 3, 4).

TTR is a protein having a molecular weight of 14 kDa composed of 127 amino acids and has a structure in which eight β-strands present inside form two antiparallel β-sheets (Non-Patent Document 5). The main production site of TTR is the liver, but it is also produced in the ventricular choroid plexus, retinal pigment epithelial cells, and spleen. TTR usually forms a tetramer with a molecular weight of 55 kDa in blood and has a stable structure, and functions mainly as a transport carrier for vitamin A-retinol binding protein complex and thyroid hormone T4 in blood and bone marrow. The blood concentration is as high as 200-400 µg/ml, but the half-life is as short as 2 days (Non-Patent Documents 2 to 6). It is known that two homologous T4 bonding sites exist in the center of the TTR tetramer, and that the tetramer structure is stabilized by the bonding of T4 (Non-Patent Document 3). As other functions, various reports have been made, such as an action to promote insulin secretion, an action to protect the brain nerve, and an action related to lipid metabolism (Non-Patent Document 2). On the other hand, in mice knocked out of the TTR gene, the concentration of retinol and thyroid hormone in the blood was lowered, but no significant changes in phenotypes such as survival rate or reproductive ability were observed (Non-Patent Document 7), so that TTR maintains actual life activity. It is unclear whether or not it is directly essential.

In the formation of amyloid of TTR, dissociation from tetramer to monomer and structural change of the monomer are very important steps (Non-Patent Document 3). Among them, it has been found that dissociation from tetramers to monomers is a rate controlling step of the reaction. On the other hand, in the process of TTR forming amyloid and depositing in tissues and causing disorders in organs throughout the body, the molecular form that exerts toxicity to the tissues has not been completely disclosed. There are reports that low molecular weight oligomers such as monomers and dimers have cytotoxicity, and TTR amyloids of 100 kDa or more on one side do not have cytotoxicity (Non-Patent Document 5), and clarification of the relationship between toxicity and molecular form is desired. .

The treatment strategies for FAP caused by TTR gene abnormalities are mainly classified into the following four types.

(1) suppress the production level of mutant TTR

(2) stabilize the TTR tetramer structure including mutant TTR

(3) inhibits amyloidization of TTR decomposed from tetramers

(4) TTR amyloid deposited on tissues is removed

Since most of the blood TTR is produced in the liver (Non-Patent Literature 2), the most common treatment among these is liver transplantation classified as (1). The delay in progression of the condition can be confirmed by performing liver transplantation, but the patient has to use immunosuppressants for life, so the burden of surgery on the donor or patient is also high. In addition, since deposition in several organs including the eyes and heart continues, there are not a few examples of worsening symptoms of these organs (Non-Patent Document 8), and there are many problems, and the development of effective treatments is desired.

As a treatment other than liver transplantation, in the strategy of (1), the treatment using siRNA or antisense oligonucleotide is in the clinical development stage, but all are mutant and suppress the production of wild-type TTR, so when used over a long period of time The safety evaluation of the product must be made carefully. In the strategy of (2), drugs that stabilize the tetramer structure by binding to the T4 binding site of the TTR tetramer are being developed. The new drug Vyndaquel ^R developed under this strategy was approved in the EU in 2011 and in Japan in 2013. As a result of a 30-month clinical trial, Vyndaqel ^R has shown the effect of delaying peripheral nerve disorders in FAP patients, but it has not reached complete inhibition of the progression of symptoms (Non-Patent Document 9). In the other strategies (3) and (4), a plurality of types of drugs are in the clinical development stage, but neither treatment can be curative therapy.

International Publication No. WO2010030203 Patent Publication No. 2010-195710

Glenner, G.G.: Amyloid deposits and amyloidosis: the beta-fibrilloses (second of two parts).: N Engl J Med, 302:1333-1343, 1980 Ando, Y. & Jono, H.: Pathogenesis and therapy for transthyretin related amyloidosis.: Rinsho Byori, 56:114-120, 2008 Yoshiki Sekijima: Molecular Mechanism of TTR Amyloid Deposition and Its Control.: Footprints of Medicine, 229:349-356, 2009 Yuko Motozaki, Shoji Yamada: Molecular Epidemiology of Familial Amyloid Polyneuropathy (FAP).: Footprints in Medicine, 229:357-362, 2009 Hou, X., Aguilar, M.I. & Small, D.H.: Transthyretin and familial amyloidotic polyneuropathy. Recent progress in understanding the molecular mechanism of neurodegeneration.: FEBS J, 274:1637-1650, 2007 Araki, S. & Ando, Y.: Transthyretin-related familial amyloidotic polyneuropathy -Progress in Kumamoto, Japan (1967-2012)-. : Proc Jpn Acad Ser B Phys Biol Sci, 86:694-706, 2010 Episkopou, V., Maeda, S., Nishiguchi, S., Shimada, K., Gaitanaris, G.A., Gottesman, M.E. & Robertson, E.J.: Disruption of the transthyretin gene results in mice with depressed levels of plasma retinol and thyroid hormone.: Proc Natl Acad Sci U S A, 90:2375-2379, 1993 Yukio Ando: Liver Transplantation and Other Treatments for Familial Amyloid Polyneuropathy (FAP): Footprints in Medicine, 229:363-368, 2009 Said, G., Grippon, S. & Kirkpatrick, P.: Tafamidis.: Nat Rev Drug Discov, 11:185-186, 2012 Goldsteins, G., Persson, H., Andersson, K., Olofsson, A., Dacklin, I., Edvinsson, A., Saraiva, M.J. & Lundgren, E.: Exposure of cryptic epitopes on transthyretin only in amyloid and in amyloidogenic mutants.: Proc Natl Acad Sci U S A, 96:3108-3113, 1999 Terazaki, H., Ando, Y., Fernandes, R., Yamamura, K., Maeda, S. & Saraiva, MJ: Immunization in familial amyloidotic polyneuropathy: counteracting deposition by immunization with a Y78F TTR mutant.: Lab Invest, 86 :23-31, 2006 Bergstroem, J., Engstroem, U., Yamashita, T., Ando, Y. & Westermark, P.: Surface exposed epitopes and structural heterogeneity of in vivo formed transthyretin amyloid fibrils. : Biochem Biophys Res Commun, 348:532-539, 2006 Matsubara, K., Mizuguchi, M. & Kawano, K.: Expression of a synthetic gene encoding human transthyretin in Escherichia coli.: Protein Expr Purif, 30:55-61, 2003. Ueda, M., Ando, Y., Hakamata, Y., Nakamura, M., Yamashita, T., Obayashi, K., Himeno, S., Inoue, S., Sato, Y., Kaneko, T., Takamune, N., Misumi, S., Shoji, S., Uchino, M. & Kobayashi, E.: A transgenic rat with the human ATTR V30M: a novel tool for analyses of ATTR metabolisms.: Biochem Biophys Res Commun, 352 :299-304, 2007. Matsubara, K., Mizuguchi, M., Igarashi, K., Shinohara, Y., Takeuchi, M., Matsuura, A., Saitoh, T., Mori, Y., Shinoda, H. & Kawano, K.: Dimeric transthyretin variant assembles into spherical neurotoxins.: Biochemistry, 44:3280-3288, 2005. Senju, S., Haruta, M., Matsumura, K., Matsunaga, Y., Fukushima, S., Ikeda, T., Takamatsu, K., Irie, A. & Nishimura, Y.: Generation of dendritic cells and Y. macrophages from human induced pluripotent stem cells aiming at cell therapy.: Gene Ther, 18:874-883, 2011.

Recently, the treatment of FAP by immunotherapy has attracted attention. In the process of forming TTR amyloid, it has been found that a new epitope (Cryptic Epitope) is exposed on the molecular surface according to the structural change of TTR (Non-Patent Document 10).

Thus, Terazaki immunized human TTR V30M transgenic mice (hTTR Tg mice), a model animal of FAP, with the TTR Y78F mutant, known as an exposed mutation of Cryptic Epitope, and evaluated the effect on TTR amyloid deposition in mouse tissues. (Non-Patent Document 11) As a result, in the group of mice immunized with the TTR Y78F mutant, a significant increase in the antibody level of the anti-TTR antibody was confirmed, and accordingly, a decrease in the amount of TTR deposition in the esophagus, stomach and intestines was recognized. . In the same test using hTTR Tg mice 18 months of age, which already showed TTR deposition, a significant decrease in the amount of TTR amyloid deposition was observed in the Y78F immune group. From the above results, it was suggested that by immunizing a mouse with a TTR mutant exposed by Cryptic Epitope, an antibody against TTR was produced in the mouse body, and as a result, the possibility of suppressing the deposition of TTR amyloid was suggested.

On the other hand, Bergstroems immunized rabbits with a peptide of the TTR 115-124 site, which is one of Cryptic Epitope, and prepared an anti-TTR115-124 polyclonal antibody (Non-Patent Document 12). When this polyclonal antibody was administered to hTTR V30M transgenic rats and the effect on TTR deposition in rat tissue was evaluated, the amount of TTR deposition in the rat intestine significantly decreased in the group administered with the polyclonal antibody. (Non-patent document 13)

As a result of these, an antibody that specifically recognizes the Cryptic Epitope of TTR specifically binds to TTR amyloid (or TTR that caused structural change constituting TTR amyloid), and inhibits formation of TTR amyloid or promotes elimination. The possibility of doing it is recognized. In other words, it is suggested that an antibody that specifically recognizes the Cryptic Epitope of TTR could be a new treatment for FAP.

Studies of anti-TTR antibodies based on this concept are reported by BIOCODEX. The company produced an amyloid-forming TTR-specific mouse monoclonal antibody AD7F6 using a TTR knockout mouse, and showed that it suppressed TTR tissue deposition using a FAP disease model Tg mouse (ATTR V30M) (patent Document 1). BIOCODEX's patent claims the amino acid sequence of a mouse antibody, and administration to humans is difficult. In addition, the presence or absence of reactivity to the V30M mutant having a tetrameric structure is not stated with respect to this antibody. In FAP patients with the V30M mutation, the V30M mutant present in the blood has a tetrameric structure, but it is thought that a part of it causes structural change after dissociation as a monomer to form amyloid. Therefore, an antibody that does not respond to the V30M mutant that has a tetrameric structure, but only responds to the amyloidized (or in the middle of amyloid formation) V30M mutant, is considered a necessary requirement for realizing a more effective and safe antibody therapy. do. Moreover, regarding the reactivity of this antibody, since only serum of the V30M carrier was used as a clinical sample, the reactivity with the tissue-depositing amyloid present in the patient's body is unclear.

Studies of anti-TTR antibodies based on the same concept have also been reported by a group at Porto University in Portugal (Non-Patent Document 10). It has been reported that mouse monoclonal antibodies mAb 39-44 and mAb 56-61 specific for TTR that caused structural change were produced, and that they react with amyloid of a V30M mutant derived from a living body. However, it is specified that these did not show an inhibitory effect on the formation of amyloid, and the possibility that they could be used for diagnosis of FAP is only mentioned.

As described above, polyclonal antibodies or monoclonal antibodies obtained by immunizing mice (or rats) with the Cryptic Epitope of TTR have been reported to inhibit the deposition of TTR. Antibodies that have binding activity and activity to inhibit fibrosis of TTR, and humanized antibodies or human antibodies suitable for administration to the human body have not been reported.

In TTR amyloidosis, the present inventors thought that after some tetrameric TTR dissociated into a monomer, the monomer TTR causes a structural change and forms amyloid. On the other hand, there is also a tetrameric TTR that functions normally. Therefore, the present inventors investigated an antibody having an activity that specifically binds to TTR that caused structural change and inhibits fibrosis of TTR. In addition, with the aim of finally targeting antibody medicine against TTR amyloidosis, the present invention was completed by repeated intensive studies on human antibodies having the above-described activities.

That is, the present invention relates to the following.

(1) A human antibody having an activity of inhibiting fibrosis of transthyretin (hereinafter referred to as "TTR").

(2) The human antibody specifically recognizing the TTR that caused the structural change in the above (1).

(3) a human antibody specifically binding to TTR amyloid in the above (1) or (2);

(4) The human antibody in any one of the above (1) to (3), which binds to TTR amyloid derived from two or more mutant TTRs.

(5) The human antibody in the above (4), wherein the mutant TTR is a TTR having a mutation selected from the group consisting of D18G, V30M, E54K, L55P, Y114C, Y116S and V122I.

(6) The human antibody according to any one of (1) to (5) above, which promotes removal of TTR amyloid.

(7) The human antibody according to any one of (1) to (6) above, which promotes macrophage phagocytosis against TTR amyloid.

(8) The human antibody according to any one of the above (1) to (7), wherein the epitope is a sequence containing the 89th position from the 79th position of the TTR.

(9) The human antibody according to the above (8), wherein the epitope is at position 89 to position 79 of the TTR.

(10) The human antibody according to any one of (1) to (9) above, which has a therapeutic effect and/or a preventive effect on TTR amyloidosis.

(11) The human antibody according to (10) above, wherein the TTR amyloidosis is familial amyloid polyneuropathy (hereinafter referred to as'FAP').

(12) The human antibody in the above (10), wherein the TTR amyloidosis is senile systemic amyloidosis (hereinafter referred to as "SSA").

(13) the antibody according to any one of (1) to (12) above, which is an antibody obtained by a phage display method;

(14) In any one of the above (1) to (13), the complementarity determining region of the H chain consisting of the following polypeptides (a) or (b), and the L chain consisting of the polypeptide of (c) or (d) Human antibody comprising the complementarity determining region of:

(a) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 1 to 3.

(b) In the amino acid sequence of SEQ ID NOs: 1 to 3, one or several amino acids are substituted, deleted, inserted, and/or added amino acid sequences, and the complementarity determining region of the H chain to TTR. Polypeptide.

(c) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 4-6.

(d) In the amino acid sequence of SEQ ID NOs: 4 to 6, one or several amino acids are substituted, deleted, inserted, and/or added to amino acid sequences, and are the complementarity determining regions of the L chain to TTR. Polypeptide.

(15) In any one of the above (1) to (13), the complementarity determining region of the H chain consisting of the following polypeptides (e) or (f), and the L chain consisting of the polypeptide of (g) or (h) Human antibody comprising the complementarity determining region of:

(e) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 7 to 9.

(f) In the amino acid sequence of SEQ ID NOs: 7 to 9, one or several amino acids are substituted, deleted, inserted, and/or added to the amino acid sequence, and become the complementarity determining region of the H chain to TTR. Polypeptide.

(g) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 10 to 12.

(h) In the amino acid sequence of SEQ ID NOs: 10 to 12, one or several amino acids are substituted, deleted, inserted, and/or added to the amino acid sequence, and become the complementarity determining region of the L chain to TTR. Polypeptide.

(16) In any one of the above (1) to (13), the H chain variable region consisting of the following (i) or (j) polypeptide, and the L chain variable region consisting of the polypeptide of (k) or (l) Human antibodies comprising:

(i) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 13.

(j) In the amino acid sequence of SEQ ID NO: 13, a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and/or added, and becomes an H chain variable region for TTR.

(k) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 14.

(l) In the amino acid sequence of SEQ ID NO: 14, a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and/or added, and serves as an L chain variable region for TTR.

(17) In any one of the above (1) to (13), the H chain variable region consisting of the following (m) or (n) polypeptides, and the L chain variable region consisting of the polypeptide of (o) or (p) Human antibody comprising

(m) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 15.

(n) In the amino acid sequence of SEQ ID NO: 15, a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and/or added, and becomes an H chain variable region for TTR.

(o) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16.

(p) In the amino acid sequence of SEQ ID NO: 16, a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and/or added, and becomes an L chain variable region for TTR.

(18) H chain variable region fragment comprising the complementarity determining region of the H chain consisting of the following polypeptides (a) or (b):

(a) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 1 to 3.

(b) In the amino acid sequence of SEQ ID NOs: 1 to 3, one or several amino acids are substituted, deleted, inserted, and/or added amino acid sequences, and the complementarity determining region of the H chain to TTR. Polypeptide.

(19) L chain variable region fragment comprising the complementarity determining region of the L chain consisting of the following (c) or (d) polypeptides:

(c) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 4-6.

(d) In the amino acid sequence of SEQ ID NOs: 4 to 6, one or several amino acids are substituted, deleted, inserted, and/or added to amino acid sequences, and are the complementarity determining regions of the L chain to TTR. Polypeptide.

(20) H chain variable region fragment consisting of the following polypeptides (i) or (j):

(i) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 13.

(j) In the amino acid sequence of SEQ ID NO: 13, a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and/or added, and becomes an H chain variable region for TTR.

(21) L chain variable region fragment consisting of the following (k) or (l) polypeptides:

(k) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 14.

(l) In the amino acid sequence of SEQ ID NO: 14, a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and/or added, and serves as an L chain variable region for TTR.

(22) H chain variable region fragment comprising the complementarity determining region of the H chain consisting of the following (e) or (f) polypeptides:

(e) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 7 to 9.

(f) In the amino acid sequence of SEQ ID NOs: 7 to 9, one or several amino acids are substituted, deleted, inserted, and/or added to the amino acid sequence, and become the complementarity determining region of the H chain to TTR. Polypeptide.

(23) L chain variable region fragment comprising the complementarity determining region of the L chain consisting of the following (g) or (h) polypeptides:

(g) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 10 to 12.

(h) In the amino acid sequence of SEQ ID NOs: 10 to 12, one or several amino acids are substituted, deleted, inserted, and/or added to the amino acid sequence, and become the complementarity determining region of the L chain to TTR. Polypeptide.

(24) H chain variable region fragment consisting of the following (m) or (n) polypeptides:

(m) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 15.

(n) In the amino acid sequence of SEQ ID NO: 15, a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and/or added, and becomes an H chain variable region for TTR.

(25) L chain variable region fragment consisting of the following (o) or (p) polypeptides:

(o) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16.

(p) In the amino acid sequence of SEQ ID NO: 16, a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and/or added, and becomes an L chain variable region for TTR.

(26) the H chain variable region fragment containing the H chain complementarity determining region described in (18) above or the H chain variable region fragment described in (20) above, and the complementarity determining region of the L chain described in (19) above. A single-chain variable region fragment comprising an L chain variable region fragment or an antibody against TTR obtained by linking the L chain variable region fragment according to (21) above.

(27) The H chain variable region fragment containing the complementarity determining region of the H chain described in (18) above, or the H chain variable region fragment described in (20) above, and/or the complementarity of the L chain described in (19) above. The human antibody according to any one of (1) to (13), which is formed by linking a human-derived constant region to the L chain variable region fragment or the L chain variable region fragment described in (21) above, comprising a crystal region, or snippet.

(28) the H chain variable region fragment containing the H chain complementarity determining region described in (22) above, or the H chain variable region fragment described in (24) above, and the complementarity determining region of the L chain described in (23) above. A single-chain variable region fragment to an antibody against TTR obtained by linking the L chain variable region fragment or the L chain variable region fragment according to (25) above.

(29) H chain variable region fragment containing the complementarity determining region of the H chain described in (22) above, or the H chain variable region fragment described in (24) above, and/or the complementarity of the L chain described in (23) above. The human antibody according to any one of (1) to (13), which is formed by linking a human-derived constant region to the L chain variable region fragment or the L chain variable region fragment described in (25) above, comprising a crystal region, or snippet.

(30) The gene encoding the antibody or fragment thereof according to any one of (1) to (29) above.

(31) A recombinant expression vector containing the gene according to (30) above.

(32) A transformant into which the gene according to (30) or the expression vector according to (31) has been introduced.

(33) A mechanism for detecting TTR amyloid containing the antibody or fragment thereof according to any one of (1) to (29) above.

(34) A reagent for detecting TTR amyloid containing the antibody or fragment thereof according to any one of (1) to (29) above.

(35) A carrier that slides to remove TTR amyloid containing the antibody or fragment thereof according to any one of (1) to (29) above.

(36) A diagnostic drug for TTR amyloidosis comprising the antibody or fragment thereof according to any one of (1) to (29) above.

(37) The diagnostic drug according to (36) above, wherein the TTR amyloidosis is FAP.

(38) The diagnostic drug according to (36) above, wherein the TTR amyloidosis is SSA.

(39) A fibrosis inhibitor of TTR comprising the antibody or fragment thereof according to any one of (1) to (29) above.

(40) A pharmaceutical composition for preventing and/or treating TTR amyloidosis comprising the antibody or fragment thereof according to any one of (1) to (29) above.

(41) The pharmaceutical composition according to (40) above, wherein the TTR amyloidosis is FAP.

(42) The pharmaceutical composition according to (40) above, wherein the TTR amyloidosis is SSA.

(43) A method for measuring the activity of inhibiting fibrosis of TTR, comprising a step of reacting mutant TTR with an evaluation sample in the presence of Na deoxycholate.

(44) In the above (43), the method of forming fibers under a neutral condition.

(45) The method according to (431) or (44) above, wherein the concentration of Na deoxycholate is 0.1% to 1%.

(46) The method according to any one of (43) to (45) above, wherein the evaluation sample is an antibody against TTR.

(47) The method according to any one of (43) to (46) above, wherein the mutant TTR is V30M TTR.

The present inventors have succeeded in creating a monoclonal antibody that specifically recognizes TTR that caused a structural change, and pioneering a path for the development of an antibody drug that enables the treatment of FAP. The antibody of the present invention effectively inhibits the formation of amyloid fibers and deposition of TTR into tissues, and further does not react with normal TTR functioning in the blood, so it is expected to be an antibody drug having excellent safety. In addition, as a mechanism of action, (1) inhibiting the formation of TTR amyloid and deposition of tissues, and (2) promoting clearance by acting on TTR amyloid deposited in tissues, that is, reducing the once accumulated amyloid. The same two effects can be expected. Such an effect could not be successful in the prior art or with prior developments, and expectation of this antibody therapy as a novel treatment strategy for TTR amyloidosis is desired.

As described above, the antibody of the present invention not only can be expected to provide a new treatment for FAP other than liver transplantation, but also has the potential as a drug for treating senile systemic amyloidosis (SSA). In TTR amyloidosis, in addition to FAP, which is caused by a mutation in the TTR gene, SSA is known, which is caused by wild-type TTR mainly causing amyloid deposition in the heart. It is also given a location in the heart of Alzheimer's disease. Amyloid deposition is also recognized in the lungs, blood vessel walls, and renal medulla. Patients are asymptomatic, often show heart symptoms (moderately progressive heart failure), and sometimes show carpal tunnel syndrome. It is said that the onset has been recognized since the 60th generation, and about 4 to 1 people over 80 years of age have it. Estimates of more than 400,000 patients have been reported in the United States alone. No effective treatment has been established for this disease. Since the antibody of the present invention has an activity of inhibiting fibrosis of wild-type TTR, it is also expected to be applied to SSA.

This antibody preparation is expected to be applied not only to FAP, but also to amyloid-forming diseases to various organs caused by TTR, and is expected to make a therapeutic contribution to many patients with these diseases who do not currently have a treatment method.

1 shows (a) chimeric 371 antibody H chain expression vector pKMA010-371-mCg1, (b) chimeric 371 antibody L chain expression vector pKMA009-371-mCλ, (c) chimeric 313 antibody H chain expression vector pKMA010-313-mCg1 , And (d) a map of the chimeric 313 antibody L chain expression vector pKMA009-313-mCλ.
2 shows the results of epitope analysis. (a) shows the analysis results for the 76-85 area of the TTR, and (b) the 84-93 area of the TTR, respectively.
3 shows the results of reaction specificity analysis using the surface plasmon resonance method. (a) shows the results of the 371M antibody, (b) the 313M antibody, (c) the Dako polyclonal antibody, and (d) the negative control antibody, respectively.
4 shows the results of the analysis of reactivity to patient serum. (a) shows the results of the 371 antibody and (b) the 313 antibody, respectively.
5A shows the results of analysis of reactivity to patient tissue (paraffin section).
5B shows the results of analysis of reactivity to patient tissues (frozen sections).
6 shows the results of reactivity analysis with TTR fibers. (a) shows the results of silver staining, (b) shows the results of western brotting in the 371M antibody, and (c) shows the results of the 313M antibody, respectively.
7 shows the results of a preliminary examination of the TTR fibrosis inhibition test.
8 shows the results of preliminary examination of the TTR fibrosis inhibition test.
9 shows the results of the TTR fibrosis inhibition test. (a) shows the results of 371 antibody and 313 antibody, and (b) shows the results of 371M antibody and 313M antibody, respectively.
10 shows the results of a macrophage phagocytosis test. (a) shows the results of untreated purified V30M, and (b) shows the results of TTR fibers, respectively.
11 shows the results of a drug efficacy evaluation test using V30M Tg rats.

The specific form of the present invention will be described below. In addition, the present invention is not limited to this.

1. Recombinant human antibody and fragment thereof of the present invention

In the present invention, in order to obtain a human antibody that specifically binds to TTR with a structural change and inhibits fibrosis of TTR, paying attention to the structural change of S112I of TTR to exist as a dimer, and selecting S112I as an antigen for antibody production. , A human antibody was prepared by the phage display method. As an antibody production technology using phage display technology, ``Phage Display of Peptides and Proteins: A Laboratory Manual Edited by Brian K. Kay et al'', ``Antibody Engineering: A PRACTICAL APPROACH Edited by J. McCAFFERTY et al'', ``ANTIBODY ENGINEERING second'' edition edited by Carl AK BORREBAECK” is illustrated. In the present invention, the fiber obtained by acid treatment of S112I is solidified on a panning plate, and then reacted with a phage library. The acid treatment may appropriately select the temperature, time, and pH at which S112I turns into fibers. The presence or absence of fibrosis may be confirmed by ThioflavinT assay. For example, a method of treating S112I at 37°C for 16 hours under the conditions of pH3.0-pH4.4 is illustrated. Then, after washing the plate to remove unbound phage, the phage bound to the target molecule is recovered and infected with E. coli. After culturing E. coli, the phage is recovered and reacted to the plate again. After performing such a series of panning cycles about 3-5 times, a phage excellent in reactivity with S112I is selected by ELISA test.

Then, the plasmid DNA derived from the phage is recovered from E. coli that produces the selected phage, and the base sequence of the VH region (or VL region) is analyzed. With respect to an expression vector containing a nucleotide sequence encoding an H chain or L chain constant region, a nucleotide sequence of the VH region or VL region is inserted to prepare an H chain or L chain expression vector. The expression vector is introduced into an appropriate host (animal cell), and a human antibody is expressed using the host.

In the present invention, two kinds of human antibodies having different CDR amino acid sequences were obtained, and improved human antibodies were prepared in which the amino acid sequence of the framework portion was mutated with respect to these antibodies (371M, 313M).

A method of analyzing an epitope for a human antibody is illustrated. Among the amino acid sequences at positions 76-93 of the human TTR, a peptide in which 1 amino acid was changed to alanine (position 91 was changed to serine because of alanine) to prepare a human TTR variant. A gene of a variant is produced by the conventional site-directed mutagenesis method, inserted into an expression vector, and then expressed and purified using an appropriate host (preferably E. coli). The modified product is subjected to SDS-PAGE electrophoresis using a conventional Western blotting method, and then reacted with the antibody to be analyzed, and the reactivity between the modified product and the antibody is detected. In the case where there is a variant whose binding property to the antibody has decreased, the modified portion may be considered as an epitope portion of the antibody. The antibody of the present invention has an epitope moiety at positions 78-89, and it is assumed that such moieties are present in a hidden moiety in the wild-type TTR tetramer. Moreover, this epitope was a novel epitope.

The above human antibodies have specific reactivity to TTR that caused structural changes, reactivity with fibrotic TTR, immunostaining of tissues derived from TTR patients, inhibitory activity of mutant TTR against fibrosis, and macrophage phagocytosis against TTR amyloid. Promotional activity and efficacy evaluation using FAP model animals may be carried out.

As a method of analyzing the specific reactivity to TTR that caused structural change, a method using the surface plasmon resonance method is exemplified. Wild-type TTR tetramers, mutant TTR (V30M, etc.) tetramers, or mutant TTR amyloids are constructed. As a method of producing a wild-type TTR tetramer or a mutant TTR tetramer, the method of Matsubara (Non-Patent Document 13) is exemplified. As a method for producing mutant TTR amyloid, the above acid treatment is illustrated. Next, these adjustments are coupled to the sensor chip. Then, by flowing the antibody to be analyzed and reacted with the sensor chip, the binding properties of the TTR and the antibody appear as a response unit (RU). In this case, it is considered that an antibody with a higher RU against the mutant TTR amyloid than that of the wild-type TTR tetramer or the RU against the mutant TTR tetramer specifically recognizes the TTR that caused the structural change. Compared with such tetrameric TTR, an antibody that specifically recognizes TTR (for example, TTR amyloid) having a tetrameric or higher molecular structure may be used as an antibody that specifically recognizes the TTR that caused the structural change.

The method of analyzing the activity of inhibiting the fibrosis of TTR is illustrated. With respect to the solution containing the TTR and the evaluation antibody, a surfactant is mixed to a final concentration of 0.01-1%, and the TTR is allowed to stand at a temperature and time to form fibers. Then, the fluorescence intensity was measured by ThioflavinT assay (excitation wavelength 440 nm, fluorescence wavelength 480 nm), and the degree of TTR fibrosis was evaluated. Examples of surfactants include benzalkonium chloride, sodium deoxycholate, Zwittergent3-16, and NP-40. In particular, deoxycholic acid is preferred. A final concentration of 0.01-1% is exemplified, but 0.1% is particularly preferred. The time and temperature are exemplified at 37° C. for 3 to 4 days, but may be changed in a timely manner according to a combination of time and temperature. This analysis method is an excellent evaluation system capable of suppressing denaturation of the evaluation antibody because it can be analyzed at a pH near neutral.

The method of analyzing the macrophage phagocytosis action on TTR amyloid is illustrated. Human iPS cells are produced from healthy adult-derived skin tissue according to the conventional method, and further, iPS cells are differentiated into macrophages according to the conventional method. TTR fibers and 5×10 ⁴ differentiated macrophages are mixed, an evaluation antibody is added, and incubated for a certain period (eg, 3 days). The remaining amount of TTR after cultivation is quantified by ELISA, and phagocytosis by macrophage is evaluated.

A method of analyzing the reactivity between the human antibody of the present invention and TTR amyloid is illustrated. The wild-type TTR or the mutant TTR is treated under an acidic condition and a time for the TTR to fibrillate to produce TTR amyloid. The time for fibrosis can be appropriately selected according to the type of pH or TTR. When a sample after acid treatment is run on Native PAGE and silver staining is performed, when a broadband is confirmed at a position higher than 60 kDa, the TTR may be used as an index of fibrosis. Then, TTR amyloid is subjected to SDS-PAGE using a conventional Western blot method, and the antibody to be analyzed is reacted and detected. Compared with TTR not subjected to acid treatment (TTR not fibrillated), an antibody having a high reactivity to TTR amyloid may be used as an antibody having a binding action to TTR amyloid.

The method of drug efficacy evaluation using FAP model animals is illustrated. Using a V30M Tg rat (Non-Patent Document 14, a transgenic rat in which the human TTR gene was mutated from valine to methionine at position 30 in the TTR amino acid sequence), a certain amount of an evaluation antibody was used (for example, 10 mg/kg), over a period of time (eg, for 6 months) and at a constant frequency (eg, once a week). After administration, an autopsy is performed, the large intestine is collected, and formalin is fixed. The fixed colon tissue is embedded in a paraffin block, and a tissue section is prepared. Tissue sections were immunostained using Polyclonal Rabbit Anti-Human Prealbumin (Dako) and HRP-labeled Goat anti-Rabbit IgG (Dako), and the degree of TTR deposition in the muscle layer of the colon was quantified and compared between groups. .

The human antibody of the present invention has an activity that inhibits fibrosis of TTR, a specific binding activity against TTR with a structural change, an effect of promoting phagocytosis of TTR amyloid by macrophages, an effect of binding activity against TTR amyloid, and an effect against FAP model animals. Have. Moreover, as a result of analyzing the epitope for the antibody of the present invention, it was present in TTR78-89. Therefore, the following human antibodies are included in the present invention.

(1) A human antibody having an activity to inhibit fibrosis of TTR.

(2) A human antibody that specifically recognizes the TTR that caused the structural change and does not recognize the functional TTR of the tetramer.

(3) Human antibody that specifically binds to TTR amyloid.

(4) Human antibody that promotes the removal of TTR amyloid.

(5) Human antibodies that promote macrophage phagocytosis against TTR amyloid.

(6) A human antibody having a therapeutic effect and/or a preventive effect on TTR amyloidosis.

(7) Human antibody using TTR78-89 as an epitope.

The antibodies of (1) to (7) above may have one characteristic, as described in (1) to (7), or may have the characteristics described in (1) to (7) together. .

In the human antibody (371M) of the present invention, the amino acid sequence and base sequence of CDR1-3 on the VH region or the VL region are shown in the following table.

Accordingly, the present invention includes human antibodies having the following characteristics in amino acid sequence.

(8) A human antibody comprising the complementarity determining region of the H chain consisting of the following polypeptides (a) or (b) and the complementarity determining region of the L chain consisting of the polypeptide of (c) or (d):

(a) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 1 to 3.

(b) In the amino acid sequence of SEQ ID NOs: 1 to 3, one or several amino acids are substituted, deleted, inserted, and/or added amino acid sequences, and the complementarity determining region of the H chain to TTR. Polypeptide.

(c) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 4-6.

(d) In the amino acid sequence of SEQ ID NOs: 4 to 6, one or several amino acids are substituted, deleted, inserted, and/or added to amino acid sequences, and are the complementarity determining regions of the L chain to TTR. Polypeptide.

In addition, the human antibody may have the characteristics described in (1) to (7).

In addition, in the human antibody (313M) of the present invention, the amino acid sequence and nucleotide sequence of CDR1-3 on the VH region or the VL region are shown in the following table.

Accordingly, the present invention includes human antibodies having the following characteristics in amino acid sequence.

(9) A human antibody comprising the complementarity determining region of the H chain consisting of the following polypeptides (e) or (f) and the complementarity determining region of the L chain consisting of the polypeptide of (g) or (h):

(e) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 7 to 9.

(f) In the amino acid sequence of SEQ ID NOs: 7 to 9, one or several amino acids are substituted, deleted, inserted, and/or added to the amino acid sequence, and become the complementarity determining region of the H chain to TTR. Polypeptide.

(g) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 10 to 12.

(h) In the amino acid sequence of SEQ ID NOs: 10 to 12, one or several amino acids are substituted, deleted, inserted, and/or added to the amino acid sequence, and become the complementarity determining region of the L chain to TTR. Polypeptide.

In addition, the human antibody may have the characteristics described in (1) to (7).

The amino acid sequence and nucleotide sequence in the VH region or VL region of the human antibody 371M are shown in the following table.

Accordingly, the present invention includes human antibodies having the following characteristics in amino acid sequence.

(10) a human antibody comprising an H chain variable region consisting of the following polypeptides (i) or (j) and an L chain variable region consisting of the polypeptide of (k) or (l):

(i) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 13.

(j) In the amino acid sequence of SEQ ID NO: 13, a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and/or added, and becomes an H chain variable region for TTR.

(k) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 14.

(l) In the amino acid sequence of SEQ ID NO: 14, a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and/or added, and serves as an L chain variable region for TTR.

In addition, the human antibody may have the characteristics described in (1) to (7).

The amino acid sequence and nucleotide sequence in the VH region or VL region of the human antibody (313M) are shown in the following table.

Accordingly, the present invention includes human antibodies having the following characteristics in amino acid sequence.

(11) a human antibody comprising an H chain variable region consisting of the following (m) or (n) polypeptides and an L chain variable region consisting of the (o) or (p) polypeptide:

(m) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 15.

(n) In the amino acid sequence of SEQ ID NO: 15, a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and/or added, and becomes an H chain variable region for TTR.

(o) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16.

(p) In the amino acid sequence of SEQ ID NO: 16, a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and/or added, and becomes an L chain variable region for TTR.

In addition, the human antibody may have the characteristics described in (1) to (7).

The present invention includes H chain variable region fragments comprising the following H chain CDRs.

(12) H chain variable region fragment comprising the complementarity determining region of the H chain consisting of the following polypeptides (a) or (b):

(a) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 1 to 3.

(b) In the amino acid sequence of SEQ ID NOs: 1 to 3, one or several amino acids are substituted, deleted, inserted, and/or added amino acid sequences, and the complementarity determining region of the H chain to TTR. Polypeptide.

(13) H chain variable region fragment comprising the complementarity determining region of the H chain consisting of the following (e) or (f) polypeptides:

(e) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 7 to 9.

(f) In the amino acid sequence of SEQ ID NOs: 7 to 9, one or several amino acids are substituted, deleted, inserted, and/or added to the amino acid sequence, and become the complementarity determining region of the H chain to TTR. Polypeptide.

The present invention includes an L chain variable region fragment comprising the following L chain CDRs.

(14) L chain variable region fragment comprising the complementarity determining region of the L chain consisting of the following (c) or (d) polypeptides:

(c) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 4-6.

(d) In the amino acid sequence of SEQ ID NOs: 4 to 6, one or several amino acids are substituted, deleted, inserted, and/or added to amino acid sequences, and are the complementarity determining regions of the L chain to TTR. Polypeptide.

(15) L chain variable region fragment comprising the complementarity determining region of the L chain consisting of the following (g) or (h) polypeptides:

(g) A polypeptide consisting of the amino acid sequence of SEQ ID NOs: 10 to 12.

(h) In the amino acid sequence of SEQ ID NOs: 10 to 12, one or several amino acids are substituted, deleted, inserted, and/or added to the amino acid sequence, and become the complementarity determining region of the L chain to TTR. Polypeptide.

The following H chain variable region fragments are included in the present invention.

(16) H chain variable region fragment consisting of the following polypeptides (i) or (j):

(i) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 13.

(j) In the amino acid sequence of SEQ ID NO: 13, a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and/or added, and becomes an H chain variable region for TTR.

(17) H chain variable region fragment consisting of the following (m) or (n) polypeptides:

(m) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 15.

(n) In the amino acid sequence of SEQ ID NO: 15, a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and/or added, and becomes an H chain variable region for TTR.

The following L chain variable region fragments are included in the present invention.

(18) L chain variable region fragment consisting of the following (k) or (l) polypeptides:

(k) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 14.

(l) In the amino acid sequence of SEQ ID NO: 14, a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and/or added, and serves as an L chain variable region for TTR.

(19) L chain variable region fragment consisting of the following (o) or (p) polypeptides:

(o) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16.

(p) In the amino acid sequence of SEQ ID NO: 16, a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and/or added, and becomes an L chain variable region for TTR.

The present invention includes the following single chain variable region fragments.

(20) the H chain variable region fragment containing the complementarity determining region of the H chain according to (12) above, or the H chain variable region fragment according to (16) above, and the complementarity determining region of the L chain according to (14) above. A single-chain variable region fragment to an antibody against TTR obtained by linking the L chain variable region fragment or the L chain variable region fragment according to (18) above.

(21) the H chain variable region fragment containing the complementarity determining region of the H chain described in (13) above, or the H chain variable region fragment described in (17) above, and the complementarity determining region of the L chain described in (15) above. A single-chain variable region fragment to an antibody against TTR obtained by linking the L chain variable region fragment or the L chain variable region fragment according to (19) above.

In the single-chain variable region fragment, when linking the H chain variable region fragment and the L chain variable region fragment, they are usually connected by a suitable peptide linker or the like. As this peptide linker, for example, any single-chain peptide consisting of 10 to 25 amino acid residues can be used.

The present invention includes antibodies or fragments thereof in which a human-derived constant region is linked to the following H chain variable region fragment and/or L chain variable region fragment.

(22) The H chain variable region fragment containing the complementarity determining region of the H chain described in (12) above, or the H chain variable region fragment described in (16) above, and/or the complementarity of the L chain described in (14) above. A human antibody or fragment thereof obtained by linking a human-derived constant region to an L chain variable region fragment containing a crystal region or the L chain variable region fragment according to (18) above.

(23) The H chain variable region fragment containing the complementarity determining region of the H chain described in (13) above, or the H chain variable region fragment described in (17) above, and/or the complementarity of the L chain described in (15) above. A human antibody or fragment thereof obtained by linking a human-derived constant region to the L chain variable region fragment or the L chain variable region fragment as described in (19) above.

The antibody or fragment thereof to which the human-derived constant region is linked may be Fab, Fab', F(ab') ₂ , scAb having at least part of the Fc portion, or scFvFc, or a complete antibody. In addition, scAb means that some domain (C domain) of the L chain or H chain constant region is bound to scFv, and scFvFc means that part of the H chain constant region (Fc region) is bound to scFv.

In addition, the antibody includes a protein structurally related to an antibody, and is an immunoglobulin. In addition, the antibody of the present invention may be of any class of IgA, IgD, IgE, IgG, or IgM. In other words, it may be a monomer, or it may be a multimer such as a dimer, a trimer, a tetramer, and a pentamer.

Here, the ``one or several amino acids are substituted, deleted, inserted, and/or added'' means substitution, deletion, insertion, and/or addition by known mutant protein preparation methods such as site-specific mutagenesis method. It means that as many amino acids as possible are substituted, deleted, inserted, and/or added. Therefore, for example, the polypeptide of (b) is a mutant peptide of the polypeptide of (a), and ``mutation'' referred to herein mainly refers to a mutation artificially introduced by a known mutant protein production method, but natural (e.g. For example, the same mutant polypeptide present in humans) may be isolated and purified.

In addition, when the antibody or fragment thereof of the present invention is used as a pharmaceutical composition (in the case of administration to humans) as described below, the ``mutation'' is performed in a range that does not cause an immune response from a human or a human-derived structure, and the detection mechanism It is not particularly limited if it is used as a diagnostic drug (if not administered to humans). In addition, when administering the antibody or fragment thereof of the present invention to humans, it is preferable to perform mutation within a range that maintains the higher-order structure of the CDR that recognizes the antigen.

Antibodies and fragments thereof of the present invention may contain additional polypeptides. When such a polypeptide is added, for example, a case in which the protein of the present invention is epitope-labeled by His, Myc, Flag, or the like is exemplified.

In addition, in order to improve stability or antibody titer to the antibody and fragment thereof of the present invention, a modifier may be incorporated. That is, the antibody and fragment thereof of the present invention may be a modified antibody. Examples of this modifier include sugar chains and polymers. When the sugar chain is modified, the sugar chain may have some kind of physiological activity, but when a simple polymer modification such as polyethylene glycol (PEG) is performed, it does not exhibit its own physiological activity. In addition, there is a possibility of suppressing absorption in the liver or improving the stability in blood by PEGylation. That is, a simple polymer such as PEG is preferable as the modifier.

In addition, modifications of the antibody of the present invention and its fragments with a modifier are carried out in the same manner as in the production of the mutant peptide described above, in the case of using as a therapeutic drug, within a range that does not cause an immune response by humans, and as a detection instrument or a diagnostic kit, etc. In the case of using, it is not particularly limited. In addition, when the antibody or fragment thereof of the present invention is administered to humans, it is preferable to modify the antigen-recognizing CDR within a range that maintains the higher order structure.

2. Gene of the present invention

The present invention includes a gene encoding the antibody of 1 or a fragment thereof. For example, a gene having the following nucleotide sequence as an open reading frame (ORF) region, and a modified gene in which a part of the nucleotide sequence has been altered are included.

(1) Base sequence containing SEQ ID NO: 1 to 3, and/or SEQ ID NO: 4 to 6

(2) Base sequence comprising SEQ ID NOs: 7 to 9, and/or SEQ ID NOs: 10 to 12

(3) SEQ ID NO: 13, and/or a base sequence comprising SEQ ID NO: 14

(4) SEQ ID NO: 15, and/or a base sequence comprising SEQ ID NO: 16

Since the above gene encodes the antibody or fragment thereof of the present invention, it can be introduced into a suitable host (for example, bacterium or yeast) to express the antibody or fragment thereof of the present invention.

In addition, the gene may include a sequence such as an untranslated region (UTR) sequence or a vector sequence (including an expression vector sequence) in addition to the nucleotide sequence encoding the antibody or fragment thereof. For example, the gene of the present invention can be amplified as desired by constituting the gene of the present invention in a vector sequence from the sequence set forth in SEQ ID NO: 13 or 14, and amplifying it in a suitable host. In addition, the gene partial sequence of the present invention can also be used for a probe.

The gene of the present invention may be used as a gene therapeutic agent in diseases involving TTR amyloid. Since this gene therapy is designed to express the antibody or fragment thereof of the present invention in the body after administration, it is also possible to form the antibody or fragment thereof in the body after ingestion of the therapeutic agent and have the same effect as that of the inhibitor. none.

3. Recombinant expression vector of the present invention

The present invention includes a recombinant expression vector comprising the gene of 2, that is, the gene encoding the antibody of 1 or a fragment thereof. For example, a recombinant expression vector into which a cDNA having a nucleotide sequence shown in SEQ ID NO: 13 or 14 has been inserted is exemplified. Plasmids, phages, cosmids, etc. can be used for the construction of the recombinant expression vector, but is not particularly limited.

The specific type of the vector is not particularly limited, and a vector capable of expression from among host cells may be appropriately selected. That is, in order to reliably express the gene according to the type of host cell, a promoter sequence may be appropriately selected, and the gene according to the present invention may be incorporated into various plasmids and the like may be used as an expression vector.

Various markers can be used to confirm whether or not the gene of the present invention has been introduced into a host cell, and whether or not it is reliably expressed in a host cell. For example, a gene deleted in a host cell is used as a marker, and a plasmid or the like containing this marker and the gene of the present invention is introduced into the host cell as an expression vector. Thereby, the gene introduction of the present invention can be confirmed from the expression of the marker gene. Alternatively, the antibody according to the present invention or a fragment thereof and a marker protein may be expressed as a fusion protein. For example, a green fluorescent protein GFP (Green Fluorescent Protein) derived from a luminescent plane jellyfish is used as a marker, and the antibody according to the present invention Alternatively, the fragment may be expressed as a GFP fusion protein.

The host cell is not particularly limited, and various conventionally known cells can be suitably used. Specifically, including human or mouse-derived cells, nematodes (Caenorhabditis elegans), African claw frogs (Xenopas laevis) oocytes, cultured cells of various mammals (rat, rabbit, pig, monkey, etc.), or yellow fruit flies, Animal cells such as insect cultured cells such as silkworm moths, bacteria such as Escherichia coli, yeasts (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), etc., but are not particularly limited.

The method of introducing the recombinant expression vector into the host cell, that is, the transformation method is not particularly limited, and conventionally known methods such as electroporation method, calcium phosphate method, liposome method, and DEAE dextran method can be suitably used.

The transformant of the present invention is a transformant into which the gene of 2, ie, the gene encoding the antibody of 1 or a fragment thereof, has been introduced. Here, "introduced a gene" means that it is introduced so that it can be expressed in a target cell (host cell) by a known genetic engineering technique (gene manipulation technique). In addition, the "transformant" means not only cells, tissues, and organs, but also animal individuals. The target animal is not particularly limited, and examples thereof include mammals such as cows, pigs, sheep, goats, rabbits, dogs, cats, mormots, hamsters, mice, and rats. In particular, rodent animals such as mice and rats can be widely used as experimental animals and condition model animals, and among them, mice have many inbreds and are equipped with technologies such as cultivation of fertilized eggs and in vitro fertilization. It is preferred as a condition model animal.

In addition, the antibody of 1 or its fragment can be produced by the transformant of the present invention produced using the recombinant expression vector of the present invention.

4. Method of using the human antibody of the present invention and its fragment

The human antibody of the present invention specifically recognizes TTR (for example, TTR amyloid) that caused structural change, inhibits fibrosis of TTR, and exerts a preventive effect against FAP. Therefore, the present invention provides a mechanism for detecting structural changes of TTR, a diagnostic drug for TTR amyloidosis (particularly FAP), a drug that inhibits fibrosis of TTR, and a drug for preventing and/or treating TTR amyloidosis (particularly FAP). Contains the composition.

The present invention includes a mechanism for detecting a structural change of TTR containing the antibody of (1) or a fragment thereof (a TTR amyloid detection mechanism). Examples of the detection mechanism of the present invention include an antibody chip or an antibody column immobilized on a base (carrier) with an antibody or fragment thereof that specifically binds to a TTR with a structure-changed structure. The detection device of the present invention may be used for detecting, for example, TTR (for example, TTR amyloid) whose structure has been changed contained in a sample such as blood or urine. In addition, it may be used for diagnostic or therapeutic purposes for determining diseases involving structural changes in TTR (TTR amyloid) and evaluating therapeutic effects.

Further, the present invention includes a carrier (TTR amyloid removal carrier) used for the purpose of removing TTR amyloid containing the antibody of (1) or a fragment thereof. The removal carrier may be prepared by binding an antibody or the like to a carrier commonly used in chromatography by a general method. The above-described removal carrier is exemplified in such a use that blood is collected from a patient with amyloidosis caused by TTR amyloid, and TTR amyloid in the blood is removed through a column filled with a carrier for removing the blood.

Further, the present invention includes a reagent for detecting TTR amyloid containing the antibody of 1 or a fragment thereof (reagent for detecting TTR amyloid). That is, when labeling immunoassays such as radioimmunoassay, enzyme immunoassay, and fluorescent immunoassay using these antibodies or fragments thereof are applied, the TTR in the test sample can be quickly and accurately qualitatively or quantitatively analyzed. have. In this labeled immunoassay, the antibody or fragment thereof can be labeled with, for example, a radioactive substance, enzyme, and/or fluorescent sample and used. In addition, since these antibodies and their fragments react specifically to TTR amyloid and exhibit an immune response, if the labeling substance is measured as an indicator for the immune response, a very small amount of TTR amyloid in the test sample can be detected with high precision. I can. Compared to bioassays, the labeled immunoassay can analyze a large number of specimens at once, and at the same time, it is good because it requires less time and effort for analysis, and the analysis is characterized by high accuracy.

The present invention includes a TTR amyloidosis diagnostic drug comprising the antibody of 1 or a fragment thereof. A method of diagnosing a disease by the diagnostic drug of the present invention is to measure the amount of TTR amyloid in a test sample (blood, body fluid, tissue, etc.), and diagnose the disease according to the measurement result. In addition, diseases include diseases caused by TTR amyloid. Examples of such diseases include senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP).

The antibody of the present invention was confirmed to have an effect of inhibiting the fibrosis of TTR. Therefore, the present invention includes a drug (fibrosis inhibitor) that inhibits fibrosis of TTR containing the antibody of 1 or a fragment thereof. The fibrosis inhibitor may contain additives acceptable as pharmaceuticals, such as one or more excipients, one or more binders, one or more disintegrants, one or more lubricants, and one or more buffers.

When the human antibody of the present invention was administered to a model animal of TTR amyloidosis, the effect was confirmed. Therefore, the present invention includes a pharmaceutical composition for preventing and/or treating TTR amyloidosis containing the antibody of 1 or a fragment thereof. The pharmaceutical composition may contain additives acceptable as pharmaceuticals, such as one or more excipients, one or more binders, one or more disintegrants, one or more lubricants, and one or more buffers.

Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to this at all. For parts using commercially available kits or reagents, experiments were conducted according to the attached protocol unless otherwise noted.

Example One

Recombination S112I TTR Preparation of refined products

Referring to the method of Matsubara (Non-Patent Document 13, Non-Patent Document 15), a recombinant TTR purified product was prepared. S112I TTR (a mutant in which the 112 th serine of human TTR was replaced with isoleucine) expression vector pQE30-hTTR(S112I)-His was transformed with E. coli strain M15, and 20 ml of LB/Ampicillin (50 μg/ml)/ It was cultured at 37°C with Kanamycin (25µg/ml). When OD600nm = 0.5, IPTG was added to a final concentration of 10 mM, and cultured overnight. The cells were recovered from the culture solution by centrifugation, and suspended in BufferA (50mM PB + 0.3M NaCl + 10mM Imidazole + 20mM 2-Mercaptoethanol). The suspension was sonicated for 15 minutes, further centrifuged, and the supernatant was collected. The supernatant was subjected to His-tag purification using Ni-NTA Agarose (QIAGEN), and the eluted fraction containing the recombinant TTR was dialyzed against _{20 mM NaHCO 3.} Recombinant TTR after dialysis was purified by gel filtration using Superdex75 (GE Healthcare) under 10 mM PB (pH7.5), and the dimeric TTR fraction was used as a recombinant TTR S112I purified product.

Example 2

human TTR Genetic Cloning

In constructing the human TTR expression vector, the human TTR gene was cloned. Human liver Marathon-Ready cDNA (Clontech) in a template, a primer set at the 5'or 3'end of the mature TTR (TTR-F2: SEQ ID NO: 17, and TTR-R: SEQ ID NO: 18), and Ex- PCR was performed using Taq (Takara). After TA cloning the PCR product into pCR2.1-TOPO, the nucleotide sequence of the human TTR gene was confirmed by sequence analysis. After confirming that the sequence was correct, pCR2.1-TOPO into which the TTR gene was inserted was treated with BamHI and HindIII, and the region containing the sequence encoding the TTR gene was cut. The cleavage sequence was introduced into pQE-30 (QIAGEN) treated with BamHI and HindIII to construct a wild-type human TTR expression vector.

Example 3

human TTR Construction of mutant expression vectors

Using the human TTR expression vector constructed in Example 2 as a template, amino acid point mutation was introduced using the site-directed mutagenesis method. Point mutation introduction of amino acids is D18G, V30M, E54K, L55P, Y114C, V122I, K76A, S77A, Y78A, W79A, K80A, A81S, L82A, G83A, I84A, S85A, P86A, F87A, H88A, E89A, H90A, A91A. It implemented about 24 types of E92A and V93A, respectively. Sequences encoding the above 24 types of TTR mutants were introduced into pQE-30, respectively.

Example 4

Recombination TTR Preparation of refined products

The wild-type TTR constructed in Examples 2 and 3, or the expression vector expressing the D18G, V30M, E54K, L55P, Y114C, Y116S, V122I TTR mutants was transformed with E. coli M15, and recombinant TTR by the method shown in Example 1. The purified product was prepared. For the TTR mutant, the tetrameric TTR fraction was used as a purified recombinant TTR product.

Example 5

Anti-human TTR Isolation of antibodies

An antibody against human TTR that caused a structural change was isolated by screening a scFv phage display library prepared using human VH and VL cDNA from mRNA derived from human B cells (eg, lymph nodes or spleen). The antibody library used is very good, containing more than 1011 different antibody molecules.

The S112I TTR mutant purified product prepared in Example 1 was diluted with 10 mM PB (pH7.5) to a concentration of 3 mg/ml, and mixed with an equivalent amount of 200 mM acetic acid buffer + 100 mM NaCl (pH 4.4) to a concentration of 1.5 mg/ml. S112I TTR fiber was prepared by preparing so as to be, and reacting in a constant temperature bath at 37°C for 16 hours. For the TTR after the reaction, the fluorescence intensity was measured by ThioflavinT assay, and it was confirmed that fibrosis was in progress. ThioflavinT assay is performed by diluting with 50mM Glycine-NaOH Buffer (pH9.5) so that ThioflavinT is 20 μM and TTR is 30-60 μg/ml, and measuring the fluorescence intensity with a spectrophotometer FP-6500 (JASCO). (Excitation wavelength 440 nm, fluorescence wavelength 480 nm).

S112I TTR fibers were solidified on a Maxisorp plate (Nunc) using a standard procedure, and scFv phage specifically binding to S112I TTR fibers were obtained using the standard procedure (Antibody Phage Display Methods and protocols Edited by Philippa M. O'Brien and Robert Aitken). The clone names of the obtained scFv-phage were named "371" and "313". The binding activity of the obtained scFv phage was evaluated by the following method.

Example 6

Recombination TTR Preparation of fibers

Seven kinds of mutant TTR purified products prepared in Example 4, D18G, V30M, E54K, L55P, Y114C, Y116S, and V122I, and wild-type TTR purified products were diluted with 10 mM PB (pH7.5) to 3 mg/ml, and an equivalent amount of 200 It was mixed with mM acetic acid buffer + 100 mM NaCl (pH3.0) to prepare a concentration of 1.5 mg/ml, and reacted overnight in a 37°C incubator to prepare TTR fibers. About the TTR after the reaction, it was confirmed that a fiber was formed by ThioflavinT assay.

Example 7

term- TTR Antibody binding activity test

The antigen-binding activity of the obtained scFv phage was evaluated by ELISA. The S112I TTR fiber prepared in Example 5 and the V30M TTR fiber prepared in Example 6 were diluted to 10 µg/ml with PBS (SIGMA), and 50 µl per well were added to Maxisorp Plate (Nunc), and at room temperature for 1 hour. TTR was solidified by incubating. Into the solidified plate, 300 µl of 1% BSA-PBS was added to each well, and the plate was blocked by incubating at room temperature for 1 hour. 100 µl of the culture supernatant of the obtained scFv phage was added to each well of the plate, and incubated at 37°C. After 1 hour, the wells were washed with PBST, and 100 μl of the detection antibody anti-M13/HRP (GE Healthcare) diluted 5000 times with 1% BSA-PBS was added to each well of the plate, and incubated at 37°C. After 1 hour, it was washed with PBST, and color was developed by adding 100 µl of TMB (SIGMA) to each well of the plate. After 30 minutes, the reaction was stopped with 1N sulfuric acid, and the color development value (O.D. 450nm/650nm) was measured with a microplate reader (Molecular Devices).

As a result, scFv371 and scFv313 had excellent binding activity to S112I TTR fibers and V30M TTR fibers.

Example 8

scFv371 And scFv313 sequence Translate

The base sequence of the VH region or VL region of the obtained scFv371 and scFv313 was confirmed. The DNA base sequence contained in both phages was determined using Big Dye Terminator V3.1 Cycle sequencingkit (Applied Biosystems).

For the VH region or VL region of scFv371 and scFv313, a sequence obtained by modifying several amino acids was designed and constructed, and a 371M antibody and a 313M antibody were obtained, respectively. CDR1 to 3 in the VH region or VL region in 371M and 313M were determined by Kabat's numbering method. The amino acid sequence and nucleotide sequence of CDR1 to 3 are shown in SEQ ID NOs: 1 to 12. In addition, the amino acid sequence and nucleotide sequence of the VH region or the VL region are shown in SEQ ID NOs: 13 to 16.

Example 9

chimera 371 antibody, chimera Expression of antibody 313

The obtained sequence of the V region of scFv371 and scFv313 was introduced into a mouse IgG1λ expression vector, and these antibodies (hereinafter, chimeric 371 antibody and chimeric 313 antibody) having a mouse constant region were expressed. Using the plasmids contained in the scFv371 and scFv313 phage obtained in Example 5 as a template, the regions containing VH and VL of scFv371 and scFv313 were amplified by PCR by the combinations shown in the following table. The combination of primers and sequence numbers are shown in Table 5.

The amplified sequence is the vector pKMA010-mCg1 (XhoI, NruI treatment) expressing the mouse Cγ1 constant region as the VH region, and the VL region vector pKMA009-mCλ (XhoI, BamHI treatment) expressing the mouse Cλ constant region. Each was introduced using an In-fusion enzyme (Clontech) (Fig. 1). The pKMA010-371/313-mCg1 vector has a VH region of scFv371 or scFv313 and a mouse Cγ1 constant region inserted downstream of the CAG promoter, and has a DHFR gene as a drug resistance gene. The pKMA009-371/313-mCλ vector is a vector in which the VL region of scFv371 or scFv313 and the mouse Cλ constant region are inserted downstream of the CAG promoter. The pKMA010-371-mCg1 vector, and the pKMA009-371-mCλ vector are generally referred to as chimeric 371 antibody expression vectors. In addition, the pKMA010-313-mCg1 vector and the pKMA009-313-mCλ vector are generally referred to as chimeric 313 antibody expression vectors.

Using the VH region or VL region of scFv371 and scFv313 as a template, a mutation was introduced using the site-directed mutagenesis method, and the 371M antibody designed in Example 8, the VH region and the VL region of the 313M antibody were prepared. And similarly to the above, expression vectors of these antibodies (hereinafter, chimeric 371M antibody and chimeric 313M antibody) having a mouse constant region were constructed. The pKMA010-371M/313M-mCg1 vector is a vector in which a 371M or 313M VH region and a mouse Cγ1 constant region are inserted downstream of the CAG promoter. Further, the pKMA009-371M/313M-mCλ vector is a vector in which a 371M or 313M VL region and a mouse Cλ constant region are inserted downstream of the CAG promoter. pKMA010-371M-mCg1 and pKMA009-371M-mCλ are generally referred to as chimeric 371M antibody expression vectors, and pKMA010-313M-mCg1 and pKMA009-313M-mCλ are generally referred to as chimeric 313M antibody expression vectors.

Next, a vector expressing a 371M antibody and a 313M antibody having a human constant region (hereinafter, a human 371M antibody and a human 313M antibody, respectively) was constructed by the following method. Using the previously constructed chimeric 371M antibody and chimeric 313M antibody expression vector as a template, regions containing 371M and 313M VH and VL were amplified by PCR by the combinations shown in the following table. The combination of primers and sequence numbers are shown in Table 6.

The amplified sequence of the VH region is the vector pKMA010-hCg1 (XhoI, BamHI-treated) expressing the human Cγ1 constant region, and the VL region is the vector pKMA009-hCL (XhoI, BamHI-treated) expressing the human Cλ constant region. Each was introduced using In-fusion enzyme (Clontech). The pKMA010-371/313-hCg1 vector has a 371M antibody or 313M antibody H chain sequence inserted downstream of the CAG promoter, and has a DHFR gene as a drug resistance gene. The pKMA009-371/313-hCL vector is a vector in which the L chain sequence of the 371M antibody or 313M antibody is inserted downstream of the CAG promoter. pKMA010-371-hCg1 and pKMA009-371-hCL are generally referred to as human 371M antibody expression vectors, and pKMA010-313-hCg1 and pKMA009-313-hCL are generally referred to as human 313M antibody expression vectors. The relationship between various vectors and insertion sequences is shown in the table below.

Vectors expressing H chain and L chain of chimeric 371, 313, 371M, 313M antibodies, and human 371M, 313M antibodies were gene-transduced into Freestyle293F cells (Invitrogen) using Neofection (Astek), 37°C, 8 Various antibodies were secreted and expressed by shaking culture at 125 rpm in an environment of% CO _2. On the fifth day of culture, the supernatant was recovered, and chromatographic purification was performed using HiTrap rProteinA FF (GE Healthcare). The eluted fraction containing both antibodies was dialyzed against PBS (SIGMA), and the chimeric 371 antibody, chimeric 313 antibody, chimeric 371M antibody, chimeric 313M antibody, human 371M antibody, and human 313M antibody were purified.

Example 10

Of 371M antibody Epitope Translate

In order to analyze the epitope of the 371M antibody in more detail, the TTR alanine-substituted mutant constructed in Example 3 was used to analyze the reactivity of the 371M antibody. The TTR mutant expression vector constructed in Example 3 was transformed with E. coli strain M15, and cultured at 37°C with 20 mL of LB/Ampicillin (50 µg/ml)/Kanamycin (25 µg/ml). When O.D. 600nm = 0.5, IPTG was added to a final concentration of 1 mM, and cultured overnight. The culture solution was centrifuged, and the precipitated fraction was solubilized by Bugbuster (Merck). The solubilized bacterial fluid was run on an 8-16% SDS-PAGE gel, and transferred from the gel to Immobilon-P (Millipore). The film was blocked by adding 2% Skimmilk-PBST to the transferred film and shaking at room temperature for 1 hour. The chimeric 371M antibody was diluted with 2% Skimmilk-PBST to a concentration of 1 µg/ml, and 10 mL was added to the membrane, followed by shaking at room temperature for 1 hour. After washing with PBST, detection antibody HRP-labeled anti-mouse IgG (H+L) (AMERICAN QUALEX INTERNATIONAL) was diluted 5000 times with 2% Skimmilk-PBST, added to the membrane, and shaken at room temperature for 1 hour. After washing with PBST, color was developed with Ez West Blue (ATTO).

As a result, as shown in Fig. 2, it was found that the 371M antibody has the 78-89 site of human TTR as an epitope.

Example 11

Analysis of reaction specificity of 371M antibody and 313M antibody

In order to analyze the reactivity of the 371M antibody and the 313M antibody with respect to the TTR tetramer, reaction specificity analysis was performed using a surface plasmon resonance method. Using Biacore2000 (GE Healthcare), WT TTR tetramer, V30M TTR tetramer, and V30M TTR fibers (prepared in Examples 4 and 6, all recombinants) were solidified to about 1000 RU in Sensorchip CM5 (GE Healthcare). The ligand was solidified with 10 mM acetic acid buffer (pH 6.0). Polyclonal Rabbit Anti-Human Prealbumin (Dako), chimeric 371M antibody, chimeric 313M antibody, and negative control antibody diluted to 10 µg/ml with HBS-EP Buffer were run for 2 minutes at 20 µl/min. After electrophoresis, dissociation was performed for 60 minutes, and regeneration was performed for 30 seconds with 10 mM Gly-NaOH (pH9.0).

As a result, as shown in Fig. 3, the 371M antibody and the 313M antibody did not react to the WT TTR and the V30M tetrameric TTR, and it was confirmed that the V30M fiber was specifically recognized ((a) and (b)). Dako's polyclonal antibody reacted strongly with WT TTR and V30M TTR, and the reactivity to V30M TTR fibers was weak (c), and the negative control antibody did not react to either TTR (d).

Example 12

Analysis of Reactivity of Antibody 371 and Antibody 313 to Patient Serum

The analysis was conducted as to whether or not antibodies 371 and 313 exhibit reactivity with serum derived from patients with FAP. It is a desirable characteristic as an antibody for FAP treatment that the administered antibody does not recognize human TTR in patient serum. Serum of a normal person and serum derived from a FAP patient having a V30M TTR mutation were taken at 2 μg/ml, and wild-type TTR fibers derived from serum subjected to acid treatment in the same manner as in Example 6, and TTR amyloid extracted from the kidney of a FAP patient. At about 4 µg/ml, 100 µl per well was added to a Maxisorp plate (Nunc), and the antigen was solidified. Into the solidified plate, 300 µl of 1% BSA-PBS was added to each well, and the plate was blocked by incubating at room temperature for 1 hour. The chimeric 371 antibody and the chimeric 313 antibody were serially diluted with 1% BSA-PBS, added by 100 µl to each well of the plate, and incubated at 37°C. After 1 hour, the wells were washed with PBST, and 100 µl of detection antibody anti-mouse IgG (H+L)/HRP (Zymed) was added to each well of the plate, and incubated at 37°C. After 1 hour, it was washed with PBST, and color was developed by adding 100 µl of TMB (SIGMA) to each well of the plate. After 30 minutes, the reaction was stopped with 1N sulfuric acid, and the color development value (O.D. 450nm) was measured with a microplate reader (Molecular Devices).

As a result, as shown in Fig. 4, antibodies 371 (a) and antibody 313 (b) showed a clear concentration-dependent reactivity to those obtained by amyloidizing TTR amyloid derived from FAP patients and wild-type TTR derived from serum by acid treatment. On the other hand, it did not show a clear reactivity to serum derived from normal subjects and patients with FAP.

Example 13

Analysis of Reactivity of 371M Antibody and 313M Antibody to Patient Tissue

Hearts of FAP patients with V30M TTR were collected and formalin fixed. The fixed heart tissue was embedded in a paraffin block, and a tissue section was prepared. The tissue sections were thinly sliced to a thickness of 4 μm and adhered to a slide glass, followed by deparaffining treatment. After washing with PBS, 0.1% periodic acid dihydrate was permeated for 10 minutes, and further washed with PBS. It was immersed in Rabbit serum (Dako) 50-fold diluted with 0.5% BSA-PBS at room temperature for 1 hour, followed by blocking treatment. After washing with PBS, the chimeric 371 antibody/chimeric 313 antibody/chimeric 371M antibody/chimeric 313M antibody/negative control antibody were diluted to 10 μg/ml with 0.5% BSA-PBS, respectively, and immersed overnight at 4°C. Made it. As a secondary antibody, HRP-labeled Rabbit anti-mouse IgG (Dako) was diluted 100 times with 0.5% BSA-PBS, and immersed at room temperature for 1 hour. Washed with PBS, and developed color using DAB. Hematoxylin staining was also performed together. In the positive control, the same treatment was performed using Polyclonal Rabbit Anti-Human Prealbumin (Dako) as the primary antibody and HRP-labeled Goat anti-Rabbit IgG (Dako) as the secondary antibody. Further, considering the possibility that TTR would be denatured and the conformational structure would change due to formalin fixation, immunostaining was performed similarly to the frozen heart tissue section of a V30M TTR-bearing FAP patient. Further, in order to confirm the presence of amyloid fibers, Congo red staining was also performed. Congo red is known to settle on amyloid fibers and cause short wavelength shifts.

As a result of these analysis, as shown in Fig. 5, either of the 371 antibody / 313 antibody / 371M antibody / 313M antibody, either of the paraffin fragment (Figure 5a) and the frozen fragment (Figure 5b), deposited on the heart of the FAP patient. It was confirmed that TTR was specifically recognized.

Example 14

Of the 371M antibody and the 313M antibody TTR Analysis of reactivity to fibers

Seven types of TTR fibers prepared in Example 6 and a wild-type TTR purified product were subjected to 1.5 µg each by 8-16% SDS-PAGE, and transferred to Immobilon-P (Millipore). The film was blocked by adding 2% Skimmilk-PBST to the transferred film and shaking at room temperature for 1 hour. The chimeric 371M antibody or the chimeric 313M antibody was diluted with 2% Skimmilk-PBST to a concentration of 1 µg/ml, and 10 mL was added to the membrane, followed by shaking at room temperature for 1 hour. Washed with PBST, the detection antibody anti-mouse IgG (H+L) was diluted 5000 times with 2% Skimmilk-PBST, and shaken at room temperature for 1 hour. After washing with PBST, color was developed with Ez West Blue (ATTO).

As a result, as shown in Fig. 6, it was found that the 371M antibody and the 313M antibody recognized various TTR fibers, and on the other hand, did not recognize wild-type TTR purified products.

Example 15

V30M TTR Construction of a fibrosis inhibitory activity measurement system

Recombinant V30M TTR was diluted with PBS(-) to a concentration of 375 µg/ml, and four types of surfactants were mixed so that the final concentrations were 0.1%, 0.01%, and 0.001%. As surfactants to be used, (1) benzalkonium chloride (Yamazen Corporation), (2) sodium deoxycholate (Nacalai Tesque), (3) Zwittergent3-16 (Carbiochem), (4) NP-40 (Wako) were used. . Standing at 37°C for 4 days, the fluorescence intensity was measured by ThioflavinT assay (excitation wavelength 440 nm, fluorescence wavelength 480 nm), and the degree of TTR fibrosis was evaluated.

The results of the ThioflavinT assay are shown in FIG. 7. When both surfactants were used at a concentration of 0.1%, fibrosis of TTR progressed. Among them, when sodium deoxycholate was used, it was found that the fibrosis of TTR proceeds the fastest. Subsequently, the optimum concentration of sodium deoxycholate was examined. Recombinant V30M TTR was diluted with PBS(-) to a concentration of 375 µg/ml, and mixed so that the concentration of sodium deoxycholate was 1%, 0.5%, 0.2%, 0.1%, and 0.01%. After standing at 37°C, 4 days and 7 days later, ThioflavinT assay was performed to evaluate the degree of TTR fibrosis.

The results are shown in FIG. 8. In the condition where the sodium deoxycholate concentration was 1%, the most remarkable fibrosis was recognized, but since fibrosis was recognized even at 0 hr (right after addition), it was considered that fibrosis was rapidly progressing excessively. On the other hand, in 0.1%, fibrosis was recognized after standing overnight or more, and when compared with 0.5%, 0.2%, and 0.01%, remarkable progression of fibrosis was recognized, which was most dependent on the treatment time. From these, it was found that 0.1% of the sodium deoxycholate concentration was optimal. Until now, there has been no report on the conditions under which the fibrosis of V30M TTR proceeds in a neutral pH environment, and fibrosis of TTR is promoted by placing TTR in an acidic environment such as pH 3.0. On the other hand, when the antibody is exposed in an acidic environment, the antibody is denatured and its activity is lost. Therefore, it was difficult to evaluate the ability of the anti-TTR antibody to inhibit TTR fibrosis. By introducing deoxycholic acid into the system this time, we newly discovered that fibrosis of V30M TTR proceeds even in a neutral environment, and succeeded in constructing a system capable of evaluating the ability of anti-TTR antibodies to inhibit fibrosis.

Example 16

Of the 371M antibody, and the 313M antibody V30M TTR Fibrosis inhibition test

V30M TTR purified product, and 371 antibody, 313 antibody, 371M antibody, 313M antibody, or negative control antibody were mixed so that the molar ratio of 10 μM: 0.01 to 2 μM (TTR: 550 μg/ml, antibody: 1.5 to 300 μg/ Ml), PBS+0.1% sodium deoxycholate, it stood at 37 degreeC for 3 days. Using the sample after standing, ThioflavinT assay (excitation wavelength 440 nm, fluorescence wavelength 480 nm) was performed, and fluorescence intensity was measured.

As a result, as shown in Fig. 9, it was found that antibodies 371, 313, 371M, and 313M have an activity of inhibiting fibrosis of V30M TTR in a dependent antibody concentration.

Example 17

Macrophage Gluttony exam

When macrophages engulf the TTR fiber, a macrophage phagocytosis ability test was performed on whether or not the 371M antibody or 313M antibody promotes its ability. This test mimics the process of removing TTR deposited in TTR patient tissues by macrophages.When the phagocytosis of macrophages is promoted by the addition of both antibodies, both antibodies promote the removal of TTR deposits in human tissues. It is expected to have activity.

Human iPS cells were produced from healthy adult-derived skin tissue according to the method described in Non-Patent Document 16, and further differentiated into macrophages (iPS-MP). 1 to 2 x 10 6 iPS-MPs were pre-incubated in a 10 cm dish in the presence of 50 ng/ml hGM-SCF and 25 pg/ml M-CSF for 24 hours. After washing with PBS, iPS-MP was incubated for 10 minutes at 37° C. in a medium containing 20 μg/ml mitomycin C, and cell proliferation was stopped, so that 5×10 ⁴ pieces/100 μl/well were in a 96 well plate. Added. V30M TTR, which had been untreated or subjected to an acid treatment for 24 hours, was diluted with a medium to be 3.2 µg/ml, and then 50 µl was added at a time. Further, PBS/human 371M antibody/human 313M antibody/negative control antibody were each diluted to 40 µg/ml, and 50 µl each was added. After culturing for 2 days at 37°C and 5% CO ₂ , the culture supernatant was recovered.

The remaining amount of TTR after cultivation was quantified by the ELISA method described below, and phagocytosis by macrophage was evaluated. 5 µl of the culture supernatant was added to a 96 wel 1 plate, and 100 µl of a coating solution (25 mM sodium carbonate buffer) was added thereto, followed by standing overnight at 4°C. After washing with PBST, 250 µl of a blocking solution (0.5% gelatin dissolved in the coating solution) was added, and incubated at room temperature for 1 hour. After washing with PBST, Polyclonal Rabbit Anti-Human Prealbumin (Dako) was diluted 1000-fold with 0.05% gelatin-PBST, 100 μl was added, and incubated at room temperature for 1 hour. After washing with PBST, HRP-labeled Goat anti-Rabbit IgG (Dako) was diluted 5000-fold with 0.05% gelatin-PBST, added each 100 µl, and incubated at room temperature for 1 hour. After washing with PBST, color development was performed for 5 minutes with 100 µl of SureBlue (KPL), and color development was stopped with 100 µl of 1M hydrochloric acid. The wavelength of 450 nm was measured with an xMARK microplate reader (Bio-Rad Laboratories).

The results are shown in FIG. 10. For untreated purified V30M, there was no statistically significant difference in the TTR residual amount in either sample (a), for TTR fibers, compared to PBS (none), statistically significant in 371M antibody and 313M antibody. A decrease in the residual amount of TTR was recognized (b), and it was found that the 371M antibody and the 313M antibody have the activity of promoting the TTR fiber phagocytosis of human iPS cell differentiation macrophages.

Example 18

V30M Tg Rat Drug efficacy evaluation test used

Using a 3-month-old V30M Tg rat (Non-Patent Document 14, a transgenic rat into which a human TTR gene mutated from valine to methionine at position 30 in the TTR amino acid sequence) was used, the chimeric 371 antibody, the chimeric 313 antibody Each 10 mg/kg or PBS was administered 26 times a week for 7 or 8 animals in each group, over 6 months of 3 to 9 months of age. After administration, autopsy was performed, the large intestine was collected, and formalin was fixed. The fixed colon tissue was embedded in a paraffin block, and a tissue section was produced. Tissue sections were immunostained using Polyclonal Rabbit Anti-Human Prealbumin (Dako) as the primary antibody and HRP-labeled Goat anti-Rabbit IgG (Dako) as the secondary antibody, and the degree of TTR deposition in the muscle layer of the colon was measured. They were quantified and compared between groups.

As a result, as shown in FIG. 11, TTR deposition was significantly suppressed in the 371 antibody administration group and the 313 antibody administration group compared to the PBS administration group.

(Industrial availability)

Because the recombinant human anti-transtyretin antibody of the present invention has excellent activity (TTR fibrosis inhibitory activity, macrophage phagocytosis promoting activity, etc.) and/or specificity (specifically recognizes TTR and TTR fibers that caused structural changes), It is useful as an effective drug against various diseases involving structural changes and fibrosis of TTR.

SEQUENCE LISTING <110> The Chemo-Sero-Therapeutic Research Institute <120> Anti-transthyretin human antibody <130> 672252 <150> JP 2014-014912 <151> 2013-01-29 <160> 41 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Homo sapiens <400> 1 Ser Tyr Ala Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Homo sapiens <400> 2 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 10 <212> PRT <213> Homo sapiens <400> 3 Gly Thr Arg Thr Asn Trp Tyr Phe Asp Leu 1 5 10 <210> 4 <211> 13 <212> PRT <213> Homo sapiens <400> 4 Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> Homo sapiens <400> 5 Ser Asn Asn Gln Arg Pro Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Homo sapiens <400> 6 Ala Ala Trp Asp Asp Ser Leu Tyr Gly Pro Val 1 5 10 <210> 7 <211> 5 <212> PRT <213> Homo sapiens <400> 7 Ser Tyr Tyr Met His 1 5 <210> 8 <211> 17 <212> PRT <213> Homo sapiens <400> 8 Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 9 <211> 13 <212> PRT <213> Homo sapiens <400> 9 Phe Gly Ser Ser Ser Arg Gly Asn Asp Ala Phe Asp Ile 1 5 10 <210> 10 <211> 11 <212> PRT <213> Homo sapiens <400> 10 Ser Gly Asp Val Leu Ala Lys Lys Tyr Ala Arg 1 5 10 <210> 11 <211> 7 <212> PRT <213> Homo sapiens <400> 11 Lys Asp Ser Glu Arg Pro Ser 1 5 <210> 12 <211> 10 <212> PRT <213> Homo sapiens <400> 12 Tyr Ser Ala Ala Asp Asn Lys Glu Ala Val 1 5 10 <210> 13 <211> 119 <212> PRT <213> Homo sapiens <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Thr Arg Thr Asn Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 14 <211> 110 <212> PRT <213> Homo sapiens <400> 14 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Arg Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Tyr Gly Pro Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 110 <210> 15 <211> 122 <212> PRT <213> Homo sapiens <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Phe Gly Ser Ser Ser Arg Gly Asn Asp Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 16 <211> 107 <212> PRT <213> Homo sapiens <400> 16 Ser Tyr Glu Leu Thr Gln Pro Ser Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Val Leu Ala Lys Lys Tyr Ala 20 25 30 Arg Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Ala Gln Val Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Tyr Ser Ala Ala Asp Asn Lys Glu Ala 85 90 95 Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu 100 105 <210> 17 <211> 27 <212> DNA <213> Artificial <220> <223> primer <400> 17 cgcggatccg gccctacggg caccggt 27 <210> 18 <211> 36 <212> DNA <213> Artificial <220> <223> primer <400> 18 cccaagcttt tatcattcct tgggattggt gacgac 36 <210> 19 <211> 48 <212> DNA <213> Artificial <220> <223> primer <400> 19 tgttgctatt ctcgagggtg tccagtgtga ggtgcagctg gtggagtc 48 <210> 20 <211> 36 <212> DNA <213> Artificial <220> <223> primer <400> 20 ggggtgtcgt tttcgctgag gagacggtga ccaggg 36 <210> 21 <211> 40 <212> DNA <213> Artificial <220> <223> primer <400> 21 gggtcccagg ctcgagtggg tcctatgagc tgacacagcc 40 <210> 22 <211> 44 <212> DNA <213> Artificial <220> <223> primer <400> 22 aagcgtattt ggatccactc acctaggacg gtcagctggg tgcc 44 <210> 23 <211> 48 <212> DNA <213> Artificial <220> <223> primer <400> 23 tgttgctatt ctcgagggtg tccagtgtca aatgcagctg gtgcagtc 48 <210> 24 <211> 39 <212> DNA <213> Artificial <220> <223> primer <400> 24 ggggtgtcgt tttcgctgaa gagacggtga ccattgtcc 39 <210> 25 <211> 48 <212> DNA <213> Artificial <220> <223> primer <400> 25 tgttgctatt ctcgagggtg tccagtgtca ggtccagctg gtacagtc 48 <210> 26 <211> 15 <212> DNA <213> Homo sapiens <400> 26 agctatgcca tgagc 15 <210> 27 <211> 51 <212> DNA <213> Homo sapiens <400> 27 gctattagtg gtagtggtgg tagcacatac tacgcagact ccgtgaaggg c 51 <210> 28 <211> 30 <212> DNA <213> Homo sapiens <400> 28 gggacccgga cgaactggta cttcgatctc 30 <210> 29 <211> 39 <212> DNA <213> Homo sapiens <400> 29 tctggaagta gatccaacat cgggagtaat actgttaac 39 <210> 30 <211> 21 <212> DNA <213> Homo sapiens <400> 30 agtaataatc agcggccctc a 21 <210> 31 <211> 33 <212> DNA <213> Homo sapiens <400> 31 gcagcatggg atgacagtct gtatggtcct gtg 33 <210> 32 <211> 15 <212> DNA <213> Homo sapiens <400> 32 agctactata tgcac 15 <210> 33 <211> 51 <212> DNA <213> Homo sapiens <400> 33 ataatcaacc ctagtggtgg tagcacaagc tacgcacaga agttccaggg c 51 <210> 34 <211> 39 <212> DNA <213> Homo sapiens <400> 34 ttcgggtctt ctagcagggg gaatgatgct tttgatatc 39 <210> 35 <211> 33 <212> DNA <213> Homo sapiens <400> 35 tcaggagatg tactggcaaa aaaatatgct cgg 33 <210> 36 <211> 21 <212> DNA <213> Homo sapiens <400> 36 aaagacagtg agcggccctc a 21 <210> 37 <211> 30 <212> DNA <213> Homo sapiens <400> 37 tactctgcgg ctgacaacaa ggaggctgtg 30 <210> 38 <211> 357 <212> DNA <213> Homo sapiens <400> 38 gaggtgcagc tggtggagtc cgggggaggc gtggtccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagggacc 300 cggacgaact ggtacttcga tctctggggc cgtggcaccc tggtcaccgt ctcctca 357 <210> 39 <211> 330 <212> DNA <213> Homo sapiens <400> 39 tcctatgagc tgacacagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg gaagtagatc caacatcggg agtaatactg ttaactggta ccaacaggtc 120 ccaggaacgg cccccaaact cctcatttat agtaataatc agcggccctc aggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tggactccag 240 tctgaggatg aggctgaata ttattgtgca gcatgggatg acagtctgta tggtcctgtg 300 ttcggaggag gcacccagct gaccgtccta 330 <210> 40 <211> 366 <212> DNA <213> Homo sapiens <400> 40 caggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180 gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagtttcggg 300 tcttctagca gggggaatga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360 tcttca 366 <210> 41 <211> 321 <212> DNA <213> Homo sapiens <400> 41 tcctatgagc tgacacagcc atcctcagtg tcagtgtctc cgggacagac agccaggatc 60 acctgctcag gagatgtact ggcaaaaaaa tatgctcggt ggttccagca gaagccaggc 120 caggcccctg tgctggtgat ttataaagac agtgagcggc cctcagggat ccctgagcga 180 ttctccggct ccagctcagg gaccacagtc accttgacca tcagcggggc ccaggttgag 240 gatgaggctg actattactg ttactctgcg gctgacaaca aggaggctgt gttcggagga 300 ggcacccagc tgaccgtcct a 321

Claims (47)

The complementarity determining region of the H chain consisting of the following (a) polypeptide and the complementarity determining region of the L chain consisting of the following (c) polypeptide, or the complementarity determining region of the H chain consisting of the following (e) polypeptide and the following ( A human antibody having the activity of inhibiting fibrosis of transthyretin (hereinafter referred to as "TTR") comprising the complementarity determining region of the L chain consisting of g) polypeptide:
(a) a polypeptide consisting of the amino acid sequence of CDRs 1 to 3 shown in SEQ ID NOs: 1 to 3;
(c) a polypeptide consisting of the amino acid sequence of CDRs 1 to 3 shown in SEQ ID NOs: 4 to 6;
(e) a polypeptide consisting of the amino acid sequence of CDRs 1 to 3 shown in SEQ ID NOs: 7 to 9;
(g) A polypeptide consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 10 to 12. The human antibody according to claim 1, which specifically recognizes TTR that caused structural change. The human antibody according to claim 1, which specifically binds to TTR amyloid. The human antibody according to claim 1, which binds to TTR amyloid derived from two or more mutant TTRs. The human antibody according to claim 4, wherein the variant TTR is a TTR having a mutation selected from the group consisting of D18G, V30M, E54K, L55P, Y114C, Y116S and V122I. The human antibody of claim 1, which promotes removal of TTR amyloid. The human antibody according to claim 1, which promotes macrophage phagocytosis against TTR amyloid. The human antibody according to claim 1, wherein the epitope is a sequence comprising positions 79 to 89 of the TTR. The human antibody of claim 8, wherein the epitope is at positions 79 to 89 of the TTR. The human antibody according to claim 1, which has a therapeutic effect, a prophylactic effect, or both on TTR amyloidosis. The human antibody according to claim 10, wherein the TTR amyloidosis is familial amyloid polyneuropathy (hereinafter referred to as'FAP'). The human antibody according to claim 10, wherein the TTR amyloidosis is senile systemic amyloidosis (hereinafter referred to as'SSA'). The human antibody according to claim 1, which is an antibody obtained by a phage display method. The human antibody according to claim 1, comprising an H chain variable region consisting of the following (i) or (j) polypeptide and an L chain variable region consisting of the following (k) or (l) polypeptide:
(i) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13;
(j) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13 in which one or several amino acid residues are substituted, deleted, inserted or added, and may be an H chain variable region for TTR;
(k) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14;
(l) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14 in which one or several amino acid residues are substituted, deleted, inserted or added, and may be an L chain variable region for TTR. The human antibody according to claim 1, comprising an H chain variable region consisting of the following (m) or (n) polypeptide and an L chain variable region consisting of the following (o) or (p) polypeptide:
(m) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15;
(n) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15 in which one or several amino acid residues are substituted, deleted, inserted or added, and may be an H chain variable region for TTR;
(o) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16;
(p) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 in which one or several amino acid residues are substituted, deleted, inserted or added, and may be an L chain variable region for TTR. The H chain variable region fragment of the human antibody according to claim 1, comprising the complementarity determining region of the H chain consisting of the following (a) polypeptide:
(a) A polypeptide consisting of the amino acid sequence of CDRs 1 to 3 shown in SEQ ID NOs: 1 to 3. The L chain variable region fragment of the human antibody according to claim 1, comprising the complementarity determining region of the L chain consisting of the following (c) polypeptide:
(c) A polypeptide consisting of the amino acid sequence of CDRs 1 to 3 shown in SEQ ID NOs: 4 to 6. The H chain variable region fragment of the human antibody according to claim 1, consisting of the following (i) or (j) polypeptide:
(i) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13;
(j) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13 in which one or several amino acid residues are substituted, deleted, inserted or added, and may be an H chain variable region for TTR. The L chain variable region fragment of the human antibody according to claim 1, consisting of the following (k) or (l) polypeptide:
(k) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14;
(l) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14 in which one or several amino acid residues are substituted, deleted, inserted or added, and may be an L chain variable region for TTR. The H chain variable region fragment of the human antibody according to claim 1, comprising the complementarity determining region of the H chain consisting of the following (e) polypeptide:
(e) A polypeptide consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 7 to 9. The L chain variable region fragment of the human antibody according to claim 1, comprising the complementarity determining region of the L chain consisting of the following (g) polypeptide:
(g) A polypeptide consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 10 to 12. The H chain variable region fragment of the human antibody according to claim 1, consisting of the following (m) or (m) polypeptide:
(m) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15,
(n) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15 in which one or several amino acid residues are substituted, deleted, inserted or added, and may be an H chain variable region for TTR. The L chain variable region fragment of the human antibody according to claim 1, consisting of the following (o) or (p) polypeptide:
(o) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16;
(p) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 in which one or several amino acid residues are substituted, deleted, inserted or added, and may be an L chain variable region for TTR. A single chain variable region fragment of an antibody against TTR formed by linking the following (A) H chain variable region fragment and the following (B) L chain variable region fragment:
(A) an H chain variable region fragment comprising a complementarity determining region of an H chain consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 1 to 3; Or an H chain variable region fragment consisting of the amino acid sequence shown in SEQ ID NO: 13; Or an H chain variable region fragment consisting of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13 to which one or several amino acid residues are substituted, deleted, inserted or added and may be an H chain variable region for TTR;
(B) an L chain variable region fragment comprising the complementarity determining region of the L chain consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 4 to 6; Or an L chain variable region fragment consisting of the amino acid sequence shown in SEQ ID NO: 14; Or an L chain variable region fragment consisting of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14 to which one or several amino acid residues have been substituted, deleted, inserted or added, and which may be the L chain variable region for TTR. The human antibody of claim 1 or an antigen thereof, which is formed by linking a human-derived constant region to the following (A) H chain variable region fragment, or the following (B) L chain variable region fragment, or both (A) and (B) below. Binding fragment:
(A) an H chain variable region fragment comprising a complementarity determining region of an H chain consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 1 to 3; Or an H chain variable region fragment consisting of the amino acid sequence shown in SEQ ID NO: 13; Or an H chain variable region fragment consisting of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13 to which one or several amino acid residues are substituted, deleted, inserted or added and may be an H chain variable region for TTR;
(B) an L chain variable region fragment comprising the complementarity determining region of the L chain consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 4 to 6; Or an L chain variable region fragment consisting of the amino acid sequence shown in SEQ ID NO: 14; Or an L chain variable region fragment consisting of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14 to which one or several amino acid residues have been substituted, deleted, inserted or added, and which may be the L chain variable region for TTR. A single chain variable region fragment of an antibody against TTR formed by linking the following (A) H chain variable region fragment and the following (B) L chain variable region fragment:
(A) an H chain variable region fragment comprising a complementarity determining region of an H chain consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 7 to 9; Or an H chain variable region fragment consisting of the amino acid sequence shown in SEQ ID NO: 15; Or an H chain variable region fragment consisting of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15 to which one or several amino acid residues have been substituted, deleted, inserted or added and may be an H chain variable region for TTR;
(B) an L chain variable region fragment comprising the complementarity determining region of the L chain consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 10 to 12; Or an L chain variable region fragment consisting of the amino acid sequence shown in SEQ ID NO: 16; Or an L chain variable region fragment consisting of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 to which one or several amino acid residues have been substituted, deleted, inserted or added, and which may be the L chain variable region for TTR. The human antibody of claim 1 or an antigen thereof, which is formed by linking a human-derived constant region to the following (A) H chain variable region fragment, or the following (B) L chain variable region fragment, or both (A) and (B) below. Binding fragment:
(A) an H chain variable region fragment comprising a complementarity determining region of an H chain consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 7 to 9; Or an H chain variable region fragment consisting of the amino acid sequence shown in SEQ ID NO: 15; Or an H chain variable region fragment consisting of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15 to which one or several amino acid residues have been substituted, deleted, inserted or added and may be an H chain variable region for TTR;
(B) an L chain variable region fragment comprising the complementarity determining region of the L chain consisting of the amino acid sequences of CDRs 1 to 3 shown in SEQ ID NOs: 10 to 12; Or an L chain variable region fragment consisting of the amino acid sequence shown in SEQ ID NO: 16; Or an L chain variable region fragment consisting of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 to which one or several amino acid residues have been substituted, deleted, inserted or added, and which may be the L chain variable region for TTR. A gene encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 27. A recombinant expression vector comprising the gene according to claim 28. A transformant into which a recombinant expression vector containing the gene according to claim 28 or the gene according to claim 28 has been introduced. A mechanism for detecting a TTR amyloid comprising the antibody according to any one of claims 1 to 27 or an antigen-binding fragment thereof. A reagent for detecting a TTR amyloid comprising the antibody according to any one of claims 1 to 27 or an antigen-binding fragment thereof. A carrier for removing TTR amyloid comprising the antibody according to any one of claims 1 to 27 or an antigen-binding fragment thereof. A diagnostic drug for TTR amyloidosis comprising the antibody according to any one of claims 1 to 27 or an antigen-binding fragment thereof,
The TTR amyloidosis is Familial Amyloidotic Polyneuropathy (FAP) or Senile Systemic Amyloidosis (SSA), a diagnostic drug. The diagnostic drug according to claim 34, wherein the TTR amyloidosis is FAP. The diagnostic drug according to claim 34, wherein the TTR amyloidosis is SSA. A fibrosis inhibitor of TTR comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, for preventing or treating familial amyloid polyneuropathy (FAP) or senile systemic amyloidosis (SSA) . A pharmaceutical composition for preventing, treating, or preventing and treating TTR amyloidosis comprising the antibody according to any one of claims 1 to 27 or an antigen-binding fragment thereof,
The TTR amyloidosis is familial amyloid polyneuropathy (FAP) or senile systemic amyloidosis (SSA), a pharmaceutical composition. The pharmaceutical composition according to claim 38, wherein the TTR amyloidosis is FAP. The pharmaceutical composition according to claim 38, wherein the TTR amyloidosis is SSA. delete delete delete delete delete delete delete

Images